Pre-implantation development in mammals involves the modulation of gene expression during cell differentiation. Although a number of gene products have been identified during early murine development, only a few appear to be specific for this period of growth and still fewer have known biological roles. However, the proteins of the zona pellucida constitute a subset of gene products that are different. We have selected these proteins for detailed studies in hopes of eventually elucidating the mechanism of the control of their expression in development. During oogenesis, the zona pellucida appears as a discrete extracellular structure that surrounds the oocyte and contains three glycoproteins designated ZP-1, ZP-2 and ZP-3. This glycocalyx (a) mediates the species-specificity of capacitated sperm binding to ovulated eggs, (b) is a major block to post-fertilization polyspermy and (c) appears to protect the growing embryo as it passes down the oviduct prior to implantation on the uterine wall. We have isolated by a novel technique intact zonae pellucidae from a murine follicle culture system which mimics in vivo granulosa cell-oocyte interactions. Using radioactive precursors we have demonstrated that all three zona proteins, ZP-1, ZP-2 and ZP-3, are sulfated glycoproteins. Incubation of the follicle culture with tunicamycin (0.5 Mug/m1), an antibiotic that blocks N-glycosylation, prevents new protein deposition in the zona. Under these same conditions overall protein synthesis is decreased only 30%. Biosynthetic studies indicate that after six days in culture the zona proteins are produced at 0.4 pg/oocyte/hour, which represents 2-3% of the total oocyte protein synthesis. This synthesis stops at or before ovulation and the extremely long half-life (greater than 100 hours) of the zona may be essential to preserve its known biological functions, all of which occur after ovulation. Recently we have isolated total ovarian RNA and demonstrated the integrity of zona mRNA by microinjection of the RNA into Xenopus laevis oocytes and subsequent immunoprecipitation. This RNA will eventually be used to make a cDNA library from which clones coding for the zona proteins will be isolated.